Embryonic exposures of lithium and homocysteine and folate protection affect lipid metabolism during mouse cardiogenesis and placentation

Reproductive Toxicology 61 (2016) 82–96

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Reproductive Toxicology journal homepage: www.elsevier.com/locate/reprotox

Embryonic exposures of lithium and homocysteine and folate protection affect lipid metabolism during mouse cardiogenesis and placentation Mingda Han a,1 , Alexei V. Evsikov b,1 , Lifeng Zhang a,2 , Rosana Lastra-Vicente a , Kersti K. Linask a,∗ a b

Dept. of Pediatrics, USF Morsani College of Medicine, USF Children’s Research Institute, St. Petersburg, FL33701, United States Dept of Molecular Medicine, USF Morsani College of Medicine, Tampa, FL, United States

a r t i c l e

i n f o

Article history: Received 22 April 2015 Received in revised form 10 March 2016 Accepted 11 March 2016 Available online 15 March 2016 Keywords: Mouse embryo Heart Placenta Gender Homocysteine Lithium Folic acid Fatty acid oxidation

a b s t r a c t Embryonic exposures can increase the risk of congenital cardiac birth defects and adult disease. The present study identifies the predominant pathways modulated by an acute embryonic mouse exposure during gastrulation to lithium or homocysteine that induces cardiac defects. High dose periconceptional folate supplementation normalized development. Microarray bioinformatic analysis of gene expression demonstrated that primarily lipid metabolism is altered after the acute exposures. The lipid-related modulation demonstrated a gender bias with male embryos showing greater number of lipid-related Gene Ontology biological processes altered than in female embryos. RT-PCR analysis demonstrated significant change of the fatty acid oxidation gene Acadm with homocysteine exposure primarily in male embryos than in female. The perturbations resulting from the exposures resulted in growth-restricted placentas with disorganized cellular lipid droplet distribution indicating lipids have a critical role in cardiac-placental abnormal development. High folate supplementation protected normal heart-placental function, gene expression and lipid localization. © 2016 The Authors. Published by Elsevier Inc. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

1. Introduction Although the vertebrate mammalian embryo is well protected in the uterus, many factors, such as drugs and maternal nutritional metabolites can cross the placenta and interfere with regulatory pathways directing both placental and embryonic development early in gestation leading to congenital anomalies and intrauterine growth restriction. Differentiating cells are especially vulnerable to exposures. In the case of cardiomyocyte specification, it is one of the earliest cell-fate decisions to occur in the embryo, taking place during gastrulation. During this same developmental period of cardiogenesis, yolk sac and placental development is ongoing extra-embryonically to facilitate the exchange of nutrients and

Abbreviations: HCy, homocysteine; Li, lithium; FA, folic acid; LD, lipid droplets. ∗ Corresponding author at: David and Janice Mason Chair in Translational Cardiology, USF Children’s Research Institute, CRI #4023, 140-7th Avenue South, St. Petersburg, FL 33701, United States. E-mail address: [email protected] (K.K. Linask). 1 These authors contributed equally to these studies. 2 Present address: Shanghai Children’s Hospital and Fudan University, China.

gases between the mother and the soon functioning embryonic cardiovascular system. That congenital heart defects (CHDs) are one of the most prevalent types of human birth defects [1], most likely relates to the cardiovascular system being the first organ-system in the embryo to develop followed closely by neurogenesis. The bilateral heart fields are specified during gastrulation, a developmental period in which we can induce a high incidence of CHDs in the avian and mouse models by exposing embryos acutely to factors altering the in utero milieu [2–4]. Extrapolating our mouse data to human pregnancy, the exposures occur between the 2nd and 3rd week of human gestation post-conception. Between the second and third week of human gestation, common cell signaling pathways have been activated for cardiomyocyte and trophoblast cell specification and differentiation leading to heart development and placenta formation, respectively. The statistic that 49% of all human pregnancies are unplanned [5], indicates the first month of gestation is a high-risk period for the early embryo because the mother may not yet be taking precautionary methods to protect embryonic development. Thus, early embryogenesis is highly at risk for adverse effects of drugs, alcohol, and other adverse exposures.

http://dx.doi.org/10.1016/j.reprotox.2016.03.039 0890-6238/© 2016 The Authors. Published by Elsevier Inc. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4. 0/).

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Our acute exposure experiments targeting gastrulation on embryonic day (E) 6.75 of mouse gestation where morning of conception is taken as E0.5, relates to our perturbing not only cardiomyocyte specification and differentiation, but also trophoblast differentiation and thus subsequent placental development [6–8]. We published previously that Wnt/␤-catenin signaling important in early cardiac and trophoblast cell differentiation is perturbed by the early exposures. Several studies of mouse embryogenesis between E4.5 to E7 indicate that the trophectodermal cells overlying the inner cell mass (ICM) and the early extra-embryonic ectoderm act as a pool of stem cells for the trophoblast lineages and respond to ICM signals, including to Wnt signaling, for their differentiation and proliferation [9,10]. That results of our mouse study exposing trophoblasts may relate to human trophoblasts as well, is shown by a previously published study from my laboratory that demonstrated that exposure of incubated human HTR-8/SVneo extravillous trophoblasts to lithium (Li+ ), homocysteine (HCy), or alcohol (ethanol, EtOH) altered gene and protein expression and decreased human trophoblast cell migration [11]. In vivo acute exposure of the embryonic mouse to these three factors induced changes in the expression of similar proteins and genes in the placentas of the same mouse embryos that displayed cardiac anomalies and growth restriction [2,3]. In our present study described here we further analyzed the effects of the two factors Li+ and HCy in the induction of cardiac birth defects and placental changes in the mouse model [3]. In the human population it has been known that women with mutations in methylenetetrahydrofolate reductase (MTHFR) in the folic acid cycle have higher than normal serum levels of HCy and are at a higher risk than in the normal population to giving birth to babies with heart defects [12,13]. The metabolite HCy, a hallmark of folic acid deficiency, appears to be a causative factor in the induction of the defects [14]. Similarly, women who must be maintained on Li+ therapy during pregnancy are at a higher risk for giving birth to babies born with cardiac defects [15]. During mouse gestation, placental abnormalities and similar cardiac, including valve, defects and changes in early cardiac gene expression occur by just a single exposure during gastrulation on E6.75 to Li+ or to an elevated level of HCy [2,3,11]. This was later shown also with acute alcohol (EtOH) exposure [16]. We also demonstrated that the adverse effects of the acute exposures are prevented by dietary supplementation of folic acid initiated periconceptionally and at a relatively high concentration [3,16]. That seemingly disparate factors all induced similar cardiac and placental abnormalities in the mouse model with the same timing of exposure and that could be prevented by folic acid supplementation, suggested that some common cell processes were being perturbed that intersected with folic acid metabolism. The present study was to define dominant common processes with a focus on HCy and Li+ exposures. Recent published data of human pregnancy indicate that a male association exists in relation to severity of congenital heart defects. Results of two studies on the prevalence of congenital heart disease at live births, one study on 5190 newborns in Shanghai using echocardiographic screening [17], and a second similar study in Germany [18], indicated a male predominance in cases of severe CHDs (e.g., hypoplastic left heart syndrome; interruption of the aortic arch, single ventricle, double outlet right ventricle, tetralogy of Fallot, among others). A female predominance was observed in cases of mild CHDs (e.g., small ventricular septal defect, mild pulmonary stenosis). These data substantiate a large US population study of sex differences in mortality in children undergoing congenital heart disease surgery where it was reported more male children underwent CHD surgery and had higher-risk procedures than female [19]. Mechanisms underlying these gender-based differences are not known. Severe CHD is associated with prematurity and low birth weight. The fetus is dependent on the placenta for a

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supply of long chain polyunsaturated fatty acids that are essential in fetal growth and development and when the supply is compromised, low birth weight can ensue. Thus, we decided to incorporate analysis of the placenta and gender aspects into our study. The objective of the present study using the mouse model was to determine other dominant pathways besides Wnt signaling that are dysregulated by Li+ and HCy in cardiac and placental tissues and that are maintained normally by folate supplementation. It is noted that both Li+ and HCy exposures significantly affected expression of genes associated with Wnt signaling. We [2] and others [20,21] have reported on that previously and did not address it here further. To define other dominant pathways, we chose to use a highthroughput method, microarrays and bioinformatics analyses of the data on exposed embryonic mouse hearts, with and without high dose folic acid dietary supplementation. For analysis of the microarray data from a relatively small sample size, we adapted an approach used in cancer research focusing on Gene Ontology (GO) categories [22] followed by Visual Annotation Display analysis [23] to obtain lists of significantly over-represented GO nodes that were then analyzed for common biological processes altered by Li+ , HCy and maintained by folic acid. For such characterization and interpretation of high throughput data, GO terms provide a controlled and hierarchical manner of categorization [24]. GO is a categorization of gene products characteristics, rather than a categorization of the gene products themselves. Thus, a high number of gene products can be grouped on the basis of their characteristics as defined by the GO classifications and aid in defining processes involved in both normal development and in disease. Using GO categorization presented here, we demonstrate that alterations within processes associated with lipid metabolism were chiefly altered and occurred differentially in male and female embryos in response to the exposures in utero and that were prevented by folic acid supplementation. We then focused on the lipid-related effects of the two acute exposures administered during early pregnancy. Validation of the bioinformatics analysis in heart and placental tissues was carried out using molecular, immuno- and histochemical techniques. We suggest that our results using the mouse model in part underlie the gender differential with severity of CHDs observed in the human population [17–19]. 2. Materials and methods 2.1. Animals 2.1.1. Inbred C57BL/6J mouse strain The C57BL/6J mice were purchased from The Jackson Laboratories, Inc., Bar Harbor, ME. It has become recognized that mouse strains can differ in their response to factors [25–27]. Thus, the mouse strain being used should be taken into consideration when comparing different studies. 2.1.2. Husbandry and breeding The adult mice were housed at an ambient temperature of 22 ◦ C with a 12 h light/dark cycle and access to food and water ad libitum. For timed matings, mature male and female mice were housed overnight and the presence of a vaginal plug the following morning was taken as evidence of mating and designated as embryonic day 0.5 (E0.5). All protocols pertaining to handling of mice were approved by the Institutional Animal Care and Use Committee (IACUC) of the USF Morsani College of Medicine. 2.1.3. Control folic acid and high folic acid diets The mouse diets were specially ordered from Harlan Laboratories (Madison, WI). The control diet provides 3.3 mg/kg as the

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baseline folic acid concentration to maintain mouse health, but is an amount that we previously demonstrated does not prevent cardiac defects. The special “high” folic acid-supplemented mouse diet contains 10.5 mg/kg folic acid and prevented the noted defects. The high folic acid adjusted diet was designed to be similar in macronutrients, fiber and micronutrients (where feasible) to the 2018 standard diet, but using refined ingredients. Folic acid, 7.2 mg/kg, was added to the base of 3.3 mg/kg in the baseline diet for a total of 10.5 mg/kg. This level of folic acid represents a supplement of 460 ng/g BW (body weight) taking into consideration metabolic body size [3]. This folic acid concentration we based on an amount used in human population clinical studies [28]. The detailed diet specifications provided by Harlan are available in the Supplemental materials.

2.1.4. Exposure to HCy or Li+ Treated pregnant mice on E6.75 were randomly allocated to receive intraperitoneally (i.p.) an injection of either a single dose of 100 ␮l of 6.25 mg/ml of lithium chloride (Li+ ), as previously determined [2], or 100 ␮l of 75 ␮M HCy [3]. Control mice received i.p. injections of physiological saline (100 ␮l of 0.9% NaCl). Based on our breeding protocol and detection of the vaginal plug, an i.p. injection at 17:30 h on E6 was determined to be optimal for exposure at E6.75. All pregnant mice were placed on the morning of conception (E0.5) either on the baseline folic acid 3.3 mg/kg chow or the high dietary folic acid chow of 10.5 mg/kg. All treatment groups and control animals were then maintained on the defined diets throughout the study. On E15.5, the heart and utero-placental circulation of the embryos were examined noninvasively in utero using Doppler ultrasonography (echo) [29]. As previously published, the Li+ /HCy i.p. injections of dams on maintenance folic acid diet induced similar cardiac valve defects and altered myocardial and umbilical artery blood flow as detected noninvasively by echocardiography [3,29]. E15.5 embryos with abnormal echo patterns [3] were chosen for further analysis to compare with the control embryos displaying normal blood flow.

2.1.5. Euthanasia Pregnant mice on E15.5 were euthanized by an excess amount of CO2 with a flow rate of 4 l/min. At cessation of cardiovascular and respiratory movements, females remained in the cage for an additional minute. Once the female was removed a cervical dislocation was performed to confirm euthanasia. Uterine horns were immediately removed and placed into a petri dish of cold PBS on ice and the embryos were removed for the different assays.

2.2. Doppler echocardiography On E15.5 the pregnant mice were sedated using 3–4% Isoflurane inhalation and the echocardiography (echo) was carried out using a Vevo 770 or Vevo 2100 system (VisualSonics of SonoSite, Inc., Toronto, Ontario, Canada) to determine which embryos within the litter were displaying cardiac abnormalities. We chose E15.5 for echo analysis, as a four-chambered heart with functional valves should normally be present [29]. The embryos were visualized and their position mapped in each uterine horn. After the echo examination, all of the E15.5 embryos within the litter were isolated. Table 1 provides the percentages of mouse embryos with normal and abnormal ultrasound patterns and the numbers of resorbed embryos obtained with the acute exposures. Echo parameters previously published are provided in the Supplementary data section. Detailed analyses and discussion of the abnormal ultrasound patterns can be found in our previous publications [3,30].

2.3. Total RNA extraction and affymetrix microarray analysis Six pregnant C57BL/6J mice were used for the microarray analysis. Control embryos and folic acid-protected embryos displaying normal heart function and placental blood flow and the Li+ /HCy exposed embryos displaying semilunar valve regurgitation and abnormal blood flow patterns were isolated. Two different embryos from the same dam’s litter served as duplicate samples for each control and experimental treatment groups; therefore 6 different groups (litters), i.e., 12 embryos were analyzed. Genotyping by RTPCR for gender of embryos was not done for microarray analysis. Thus, a total of 12 embryonic E15.5 micro-dissected heart samples were analyzed by microarrays for changes in gene expression, i.e., over a week after the acute exposures were administered on E6.75. In summary, the treatment groups included embryos that received: (1) high dietary folic acid (FA) only (10.5 mg/kg body weight), with 0.9% saline by i.p., and had normal echos; (2) Li+ in saline by i.p., injection, maintenance diet FA (3.3 mg/kg body weight), and had abnormal echos; (3) Li+ in saline by i.p., injection, with high dietary FA supplementation, normal echos; (4) HCy in saline by i.p., maintenance FA diet, abnormal echos; (5) HCy in saline by i.p., with high dietary FA supplementation, normal echos; or (6) control 0.9% saline by i.p., maintenance FA diet, with normal echos. Because the outflow (OFT) and right ventricular (RV) regions were most affected with our timing of acute exposure, these regions only were microdissected together and total RNA was extracted. The left ventricles were not used. Total RNA was extracted using the RNA Micro Kit (Qiagen, Valencia, CA) and was sent to The Moffitt Cancer Center Microarray Facility that carried out the microarray hybridization. The samples were processed in one batch. Total RNA (100 ng) was obtained per tissue sample. To overcome tissue limitations, the WT-Ovation Pico RNA Amplification System (NuGEN Technologies, Inc., San Carlos, CA) was used to prepare sufficient quantities of cDNA for microarray analysis. Amplification starts at the 3 end as well as randomly throughout the whole transcriptome in the sample, thus decreasing 3 bias upon amplification. The amplified cDNA was used as input for hybridization to Affymetrix GeneChip Mouse Genome 430 2.0 arrays (Affymetrix, Inc., Santa Clara, CA). Hybridization, staining, and scanning of the chips was performed as outlined in the Affymetrix technical manual. The mouse 430 2.0 arrays contain over 45,000 probe sets designed from GenBank, dbEST, and RefSeq sequences clustered based on build 107 of the UniGene database. The clusters were further refined by comparison to the publicly available draft assembly of the mouse genome. An estimated 39,000 distinct transcripts are detected including over 34,000 well-substantiated mouse genes. Each gene is represented by a series of oligonucleotides that are identical to sequence in the gene and oligonucleotides that contain a homomeric (base transversion) mismatch at the central base position of the oligomer used for measuring cross hybridization. Scanned output files were visually inspected for hybridization artifacts and then analyzed using Affymetrix GeneChip Operating Software (GCOS). Signal intensity was scaled to an average intensity of 500 prior to comparison analysis. Using the default settings, GCOS software identifies the increased and decreased genes between any two samples with a statistical algorithm that assesses the behavior of 11 different oligonucleotide probes designed to detect the same gene [31]. Probe sets that yielded a change pvalue less than 0.002 were identified as changed (increased or decreased) and those that yielded a p-value between 0.002 and 0.002667 were identified as marginally changed. A gene was identified as consistently changed if it was identified as changed in all replicate experiments by the software. Affymetrix CEL files were analyzed using MAS5 algorithm [31,32] of Expression Console Suite (Affymetrix).

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Table 1 Comparison of Percentages of Normal and Abnormal Embryos and Numbers of Resorptions. Treatment

Litter #

Normal%

Abnormal%

Total Embryos #

Resorption #

Folate Conc

Controla High Folatea Hcysb Lib HCys + Folate Li + Folate

27 9 23 21 7 8

100 100 53.7 39.8 100 97.6

0 0 46.3 60.2 0 2.4

147 58 119 108 40 41

0 0 41 21 0 1

3.3 mg/kg 10.5 mg/kg 3.3 mg/kg 3.3 mg/kg 10.5 mg/kg 10.5 mg/kg

a b

Control animals, i.e., either on maintenance FA diet or the high FA diet with i.p. injections of physiological saline. Pregnant animals receiving i.p., injections of the specified substance with maintenance diet.

2.4. Bioinformatic analysis As indicated above we had six treatment groups (n = 6), i.e., 6 pregnant C57BL/6J mice, including the control groups. In each treatment group we analyzed two embryos from each litter. At this point of our study we were not addressing gender, but only dominant biological processes being altered and genotyping had not been done. Because developing embryos have gender biases in gene expression [33], our initial quality control of the data and gender was performed using expression values for X-inactivation transcript Xist (probesets 1427262 at, 1427263 at and 1436936 s at; present in females, absent in males) and Y-chromosome linked gene Ddx3y (1426438 at, 1426439 at and 1452077 at; present in males; absent in females). According to these probesets, our microarray data of embryos that had been picked randomly and according to echo analysis of cardiac function, the embryos represented 6 male and 6 female embryos: Control 1 male 1 female High Folate treatment 1 male 1 female Li+ treatment 1 male 1 female HCy treatment 1 male 1 female Li+ + High Folate treatment 0 male 2 female HCy + High Folate treatment 2 male 0 female Because gender differences in gene expression between arrays contributed the largest variance among data, we devised a strategy for data analysis with a low number of replicates. The strategy used is adapted from analysis done in cancer research [22]. Many clinical data are being reanalyzed using this approach because of extreme noise between individual patient samples. Focusing on GO terms (or pathways, or other types of gene function groupings) rather than individual genes, clarifies the noise. Thus to increase the sampling power of our microarray data, we tested dysregulation of expression in the groups of genes identified by the Gene Ontology (GO) categories. For this, we performed pairwise analysis for all 12 samples individually in which each probeset was assigned as “present”, “downregulated”, “upregulated” or “inconsistent” depending on the ratio of the signals between samples, and consistency of these ratios among probesets representing the same gene. Next, nonparametric analysis of these data was done using Visual Annotation Display (VLAD) that performs hypergeometric distribution of GO annotations for a given set of genes (i.e., up- or downregulated) vs. “universal set of genes (i.e., all genes ‘present’ in the samples) [23]. A p value p < 0.001 was considered of statistical significance. The outputs of each VLAD comparisons, which are the lists of significantly over-represented GO nodes, were subsequently analyzed for commonality. After obtaining the common GO categories significantly over-represented among the twelve embryos, we then sorted out the common category list according to gender of the embryos. We avoided the use of False Discovery Rate (FDR) corrections such as Benjamini-Hochberg because these methods are not suitable for FDR estimation in GO enrichment [34]. Validation of the bioinformatics results was then carried out using immuno-, including histochemical, molecular, and cellular approaches described below.

The Affymetrix microarray CEL files are deposited into the Gene Expression Omnibus (GEO), the public repository of microarray data.

2.5. RT-PCR analysis of effects of exposure and diet on lipid gene expression The gene expression changes for Acadm, Acadl, and Crt were analyzed by RT-PCR of E15.5 total hearts or placental tissues, as defined. Data was normalized in each case to the internal control ␤-actin. Relative quantitation is shown using densitometric readings from scans of the gels. Average densitometric readings were obtained. We show standard deviations, but it is to be noted that not all embryos within a litter in utero are at the same time-point of development at time of exposure and embryos analyzed were from different litters. Thus intra- and inter-litter variability led to expected variations in quantitation. We were primarily interested in determining whether patterns of changes in the defined biological processes can be observed. Our initial validation studies did not address gender, but only lipid-related changes as based on our microarray data of GO categories being modulated and on publications from other studies [35]. After sorting the bioinformatics data according to gender, we later included gender in the validation studies as related to Acadm gene described here. For gender-associated gene modulation in the second part of the study, we analyzed placental tissue from four embryos of each gender (specified by genotyping tissue from the posterior half of each embryo) for the specific exposure group having the same type of cardiac defects, specifically semilunar valve regurgitation and abnormal myocardial function and umbilical artery blood flow. The embryos were from different litters. The two lipid-related genes that we chose to analyze were based on genes that over-lapped in several of the yellow highlighted GO categories involving lipid metabolism: Specifically, the two fatty acid oxidation genes acyl-CoA-dehydrogenases -long length chain (Acadl) and –medium length chain (Acadm) overlapped in multiple categories affected by Li+ and HCy, transgenic mice for these genes had been generated, and those mice were reported to have heart-related problems [36,37]. For comparison we also analyzed calreticulin (Crt), a gene that is not directly involved in lipid metabolism and codes for a Ca + +-binding storage protein and chaperone in the endoplasmic reticulum, important in maintenance of cellular calcium homeostasis, and has a role in embryo implantation in mice [38]. Primer sequences are given in Supplemental Table I.

2.5.1. Statistics ANOVA (analysis of variance) was used to test the null hypothesis and statistical significance taking into account gender and the various treatment groups. This was followed by the two-tailed ttest to address statistical significance for the specific gender in comparison to control.

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2.6. Embryo gender determination for lipid-related gene expression analysis DNA was extracted from posterior halves of the E15.5 embryos that were chosen for sex determination according to published PCR-based methodology [39]. The reaction includes primer pairs for Sry [40] on the Y chromosome and Myog (myogenin) [39] that generate a male specific band of 380 bp and an internal control band of 245 bp, respectively. Primer sequences are provided in Supplemental Table I.

brown HRP signal present in two sections of that specific region and tissue, as compared to the total area of the delineated regions. This is defined as a percentage for each region analyzed. For the heart for each treatment, we quantified the right and left ventricular myocardium, including the adjacent trabecular region, and the interventricular septum (IVS). For the placenta we quantified a region of the maternal decidua/syncytiotrophoblasts, as well as a uniform region of the labyrinth. The sections were all immunostained at the same time following the same protocol and timing for staining, and using the same reagents.

2.7. Oil Red O (ORO) staining for lipid droplet distribution 3. Results ORO, a fat-soluble dye, was used to detect neutral lipids (triglycerides, diacylglycerols, and cholesterol esters) in cryosectioned E15.5 cardiac and placental tissues. A published staining methodology was followed that provides an estimate of tissue lipid content and localization [41]. Lipid droplets stain red and nuclei blue. Polar lipids (phospholipids, sphingolipids and ceramides) are not stained [42]. Stereoscopic quantitation of lipid droplets after exposures was not done because of disorganized placental development and uneven distribution of ORO in the labyrinth and maternal decidua of experimentally exposed tissue. Analysis of lipid droplet organization within tissues was done in a blinded manner by coding of the tissue sections of the different treatments and was carried out independently by the investigators and compared. We did not quantify numbers of lipid droplets in the tissues, because not all embryos within a litter in utero are at the same time-point of development at time of exposure and embryos analyzed were from different litters. Thus intra- and inter-litter variability was expected to lead to variations in quantitation. Also in the placental labyrinth layer there was variability in the extent of regions devoid of droplets right next to regions with droplets and intermediate zones. Quantitation would not show this. We thus chose to show representative sections of the pattern of ORO localization of the heart and placenta without quantitation. Only ORO images of heart sections were similarly enhanced in parallel using “autolevels” subroutine using Photoshop software (Adobe Systems, Inc., San Jose, CA) to bring out red color of small droplet staining relative to background and for easier observation of the organization of the staining patterns. Pattern of lipid droplets in the placenta was more distinctly noticeable and Photoshop enhancement was not done. 2.8. Immunohistochemistry and microscopy Li+ -, HCy-, and control, NaCl-exposed, embryos were fixed in 4% paraformaldehyde in PBS, paraffin embedded, and sectioned. Embryos were obtained from at least three different litters that had undergone same exposures. Duplicate sections of the tissues from the different treatments (control, experimental exposure, and high folate dietary protected tissue) were placed on the same slide and immunostaining was carried out at the same time for comparison of signal. Sections were immunostained with MCAD antibodies (Sigma, St. Louis, MO) for the protein product of Acadm gene expression. Primary antibody localization was visualized with horseradish peroxidase or fluorescent Cy-3 conjugated-secondary antibodies (Vector Laboratories, Inc., Burlingame, CA). At the end hematoxylin counterstain was used for general visualization of tissue. Localization of protein expression was analyzed with a Nikon Optiphot II phase microscope (Nikon Instruments, Inc., Melville, NY). Digitized images were obtained with a Nikon DS-L2 Camera unit. Quantification of horseradish peroxidase (HRP) brown staining in the tissues that is shown in the figures was obtained using Definiens Tissue Studio 4.0 software (Defineins AG, Munich, Germany). The score is based on the average number of pixels of

3.1. Bioinformatic analysis of microarray data The common significantly over-represented GO nodes from Li and HCy exposures based on the six treatment groups or 12 embryos (Fig. 1A and B) related to cell processes associated with lipid metabolism and fatty acid oxidation (Fig. 1A, orange highlighted categories). The following six genes involved in fatty acid oxidation were commonly represented in many of the highlighted categories: acyl CoA dehydrogenase 11 (Acad11), acyl CoA dehydrogenase long chain (Acadl), acyl CoA dehydrogenase medium chain (Acadm), acyl CoA dehydrogenase short chain (Acads), acyl CoA dehydrogenase very long chain (Acadvl), and electron transferring flavoprotein dehydrogenase (Etfdh). All of the protein products of these genes localize to the mitochondria. Our initial validation studies of effects on lipid metabolism focused on lipid droplet patterns and their modulation with exposures and diet. At the time these earlier studies were done we were not anticipating that gender may have a role. We later addressed possible gender association by re-analyzing Acadm expression in response to HCy and Li+ (see Figs. 5–7), because recent literature indicated (i) that fetal gender is associated with lipid metabolism and (ii) that gender is also associated with severity of heart defects; (iii) Acadm protein product (MCAD protein) deficiency is the most common inherited disorder of mitochondrial fatty acid ␤-oxidation in humans; and (iv) the Acadm gene was among the commonly represented genes in many of the orange highlighted categories shown in Fig. 1A. When we analyzed the list of common GO categories in Fig. 1A by comparing separately the two groups that were represented by only one gender, i.e., the lithium-exposed and folate-protected group had only two female embryos and the homocysteineexposed and folate–protected group was made up of two male embryos (shown in Fig. 1B), more perturbation was observed in the male embryonic heart transcriptome than in the female and a high number of these categories related to lipid metabolism. Overall in Fig. 1A and B male embryos showed a higher number of significant changes in the expression of genes in lipid-related categories than the female embryos. Convention of yellow cells signifying up–regulation and blue signifying down-regulation was used here. Comparing the last column in Fig. 1A of the female control groups, fol f1 vs cont f1 (i.e., the high folic acid- supplemented female embryo versus control maintenance diet female embryo) and the last column of the male control group, fol m1 vs cont m1 (high folic acid- supplemented male embryo versus control maintenance diet male embryo), it is noteworthy that although the high folic acid supplementation on its own did show some modulation of some lipid-related cellular processes, as for example ATP synthesis and energy coupled proton transport, most categories were not altered. In focusing on the orange-highlighted lipid-metabolism categories, in this small sample size in both the high folic acid diet versus maintenance diet control groups, mostly nonsignificant (NS) differences were observed for both genders (last column for each gender). This was confirmatory to our observations and ultrasound

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Fig. 1. Bioinformatic analysis generated lists of dominant changes in Gene Ontology (GO) Specific Biological Processes in embryonic hearts after exposure to Li+ or to HCy, with and without folic acid supplementation. Lists shows embryos sorted according to gender with a comparison of treated female embryos compared to control female embryos highlighted in red at top; comparison of male embryos in light blue on top right. Lipid-related GO categories (on left) are highlighted in orange. The convention used here was highlighting cells in yellow for the up-regulated processes and blue for down. Fig. 1A. Bioinformatics analysis of microarray data on Li+ - and HCy-exposed embryonic hearts, with and without high dose folic acid supplementation are contrasted with both genders for each specified category. Fig. 1B. List of overlapping GO categories with folic acid protection. In Fig. 1B folic acid protection with Li+ exposure group consisted only of female embryos (red); folic acid protection with HCy exposure group (light blue highlighting at top) consisted only of male embyros. Orange highlights GO categories overlapping with same categories as in Fig. 1A. Notable differences in response were noted. Abbreviations: f, female; m, male; vs versus; lith, lithium exposure; hcys, homocysteine exposure; lith fol, lithium exposure with high folate supplementation; hcys fol, homocysteine exposure with high folate supplementation; NS, changes not significant.

3.2. Analysis of ORO localization of neutral lipid droplet distribution in the heart and placenta

the HCy/Li+ exposures on lipid metabolism by using Oil Red O (ORO) staining for neutral lipid droplets. For packaging, cells convert lipids into neutral lipids and deposit them into intracellular organelles (termed lipid droplets or adiposomes). These droplets are important in maintaining cell homeostasis. Their accumulation in cells is linked in common pathologies seen with obesity and diabetes [44]. Previous studies linked Mthfr deficiency and low dietary folic acid in the transgenic Mthfr model also with fetal loss, intrauterine growth restriction and placental abnormalities, as well as with heart defects [45,46]. Because our exposed embryos displayed abnormal heart physiology, were growth restricted with smaller placentas, and displayed altered umbilical artery blood flow, we included analysis of the placentas. Thus, we defined whether neutral lipid synthesis and distribution were altered in the fetal E15.5 four-chambered heart (Fig. 2) or in the same embryo’s placenta (Fig. 3). In both tissues we observed changes in neutral lipid localization with more severe alterations occurring in placental tissue.

The bioinformatics analysis of the microarray data indicated that proteins involved in fatty acid ␤-oxidation and lipid metabolism are modulated with folic acid deficiency. We next evaluated effects of

3.2.1. ORO cardiac localization Lithium we reported had more severe effects on heart function than HCy [3]. This can be seen here also in that the Li+ −exposed

data that even though the high folic acid supplementation may alter gene expression within some categories, these changes maintained normal embryonic development of the heart and its function, as did the health maintenance diet with low folic acid levels. It is only with the HCy- and Li+ -exposures that significant differences in lipid metabolism-related categories appeared and often were differentially regulated by gender. It is noted that although 8 mg/kg folic acid was reported to have adverse embryonic effects in the mouse strain 129S1/SvlmJmice [43], we did not see adverse effects using 10.5 mg/kg folic acid with the C57BL/6J strain. We did not address folic acid deficiency directly, as there have been a multitude of studies indicating that low or no dietary folic acid leads to poor embryonic, including heart, development.

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Fig. 2. Oil Red O staining of neutral lipid droplet distribution (red droplets, see black arrows) in control cardiac trabeculae in left column (Fig. 2A) and in the right myocardial wall in right column (Fig. 2B), and after experimental conditions of lithium exposure (Li+ , Fig. 2C and D, respectively); Li+ with high folate (FA) supplementation (Fig. 2E, F); HCy exposure (Fig. 2G and H, respectively) and HCy with high FA supplementation (Fig. 2I and J, respectively). These ORO images of heart sections were all similarly enhanced in parallel for lipid droplet staining using “autolevels” subroutine in Photoshop.

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Fig. 3. Oil Red O staining of neutral lipid droplet distribution in labyrinth layer (Laby) and maternal deciduas (mat dec) in control (Con) placentas (Fig. 3A–F); homocysteine (HCy) exposed (Fig. 3E and H); in lithium (Li+ ) exposed placentas (Fig. 3I–L); and folic acid (FA) protected placentas (Fig. 3M–P). Boxed in labyrinth regions in 3E and I are shown at higher magnification in F,G for HCy exposure and J,K for Li+ . Magnification bars (A,B) = 250 ␮m; for other panels, bars = 100 ␮m.

Fig. 4. Modulation of Acyl CoA Dehydrogenase Gene expression in Embryonic Heart using RT-PCR. Fig. 4A: Gels shown depict Acyl CoA Dehydrogenase Acadm and Acadl gene expression in E15.5 cardiac tissue after HCy and Li+ acute exposures administered during gastrulation. Lower signal in each lane is the internal control ␤-actin. Panels 4B–G are the graphs of densitometric scan averages with standard deviations using three mouse embryos/exposure for Acadm and Acadl expression with HCys (4B and C) and Li+ (4D and E) treatments. No statistically significant changes were apparent when gender was not taken into account. Abbreviations: M, marker lane; C, control tissue; Ex, experimentally exposed tissue; FA high folic acid supplementation before exposure.

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Fig. 5. Gender-associated changes in MCAD protein expression in embryonic heart tissue on E15.5 after HCy and Li+ exposures were administered during gastrulation. Arrows point to some regions of localization. Control male (Fig. 5A) and control female (Fig. 5D) hearts show similar localization. In general a higher level of expression was apparent in the right ventricular myocardium (RV) than left (LV) and in the adjacent region of trabeculation (arrow) and in the interventricular septum (IVS; arrow). HCy exposure greatly elevated MCAD expression in male embryonic hearts (Fig. 5B) in contrast to the female (Fig. 5E; see graph in 5G). Li+ exposure showed little MCAD staining in hearts (arrows), with a slightly enhanced elevation apparent primarily in female hearts (cf. Fig. 5C and F; see graph in 5H). Inset in G shows delineated regions. Magnification bar = 500 ␮m for all panels.

hearts have thinner myocardial walls than HCy-exposed and lipid deposition was affected by the exposure. In the heart, trabeculae (Fig. 2A) and the ventricular myocardium (Fig. 2B) displayed OROstained lipid droplets (LDs), but generally at a detectably lower level than in the placental tissue. In the acute Li+ -exposed heart (Fig. 2C,D) neutral lipids were decreased and returned to control levels with folic acid supplementation (Fig. 2E,F). HCy acute exposure did not alter ORO staining in the trabeculae (Fig. 2G). The ORO staining was noticeably prevalent in the epicardial layer (Fig. 2H). Dietary high folic acid only slightly altered the exposure pattern of cardiac neutral lipid deposition (Fig. 2I and J) with HCy exposure. Pattern of ORO localization in the heart was observed to be similar among embryos from different litters of the treatment groups.

3.2.2. ORO placental localization After Li+ and HCy exposures, more significant changes in neutral lipid synthesis and organization were noted in the placenta than

in the heart and were localized to the labyrinth and the maternal decidua regions (Fig. 3A). The fetal side of the placenta did not show lipid localization in the control tissue (Fig. 3B) or after exposures (not shown). ORO localization in control tissue is at a high level in the labyrinth layer and displays a characteristic organization (Fig. 3C). The red ORO lipid droplets are organized in rows in cells along the villi. In the maternal decidua there is a high localization in the region close to the syncytiotrophoblasts (Sy Tro; 3A,D), but little staining is seen in association with the syncytiotrophoblasts themselves. With HCy exposure a patch-like neutral LD localization was evident in the labyrinth (compare Fig. 3E, E, and G). In HCy exposed villi, lipid droplets remain more organized (Fig. 3F, G) when compared with Li+ -exposed (Fig. 3J, K). Little or no ORO localization, however, was evident in large areas of the HCy-exposed labyrinth (Fig. 3F). A similar result of patch-like localization was observed in the maternal decidua layers (Fig. 3H). With Li+ exposure, the relatively uniform and organized localization within the labyrinth

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layer is lost (Fig. 3I–K). After the early acute exposures, the ORO localization and villi now appear unorganized and the ORO staining is seen in patches within the labyrinth. There are regions that are almost completely devoid of ORO deposition in the labyrinth (Fig. 3J), while a neighboring region may show intense staining (Fig. 3K). Lithium induced similar patch-like expression in a thin maternal decidua layer (Fig. 3L). Folic acid supplementation with both exposures enabled more normal development and organization of the labyrinth (Fig. 3M, N) and maternal decidua (Fig. 3O, P). Although effects of Li+ were not completely prevented by folic acid in the maternal decidua, the labyrinth development was normalized (3L). In summary, placentas of embryos from the different litters and exposed to Li+ or to HCys displayed the greatest decrease in ORO LD localization and organization in the labyrinth and maternal decidua layers. Variability related to extent of areas that were completely devoid of lipid droplets. In some placentas, the labyrinth displayed little LD localization. Disorganization of the villi was generally noted with exposure. Variability in the thickness of the maternal decidua was also noted and specifically with lithium exposure and with folic acid supplementation with lithium exposure. In all placentas, the fetal side of the placenta did not show lipid localization. 3.3. Changes in fatty acid oxidation genes with lithium/homocysteine exposure To define possible changes in lipid metabolism further, we analyzed two genes involved in fatty-acid oxidation, Acyl CoA dehydrogenase medium chain (Acadm) and Acyl CoA dehydrogenase long chain (Acadl). Both genes appeared multiple times in the lipidrelated GO categories defined in the microarray analysis. These genes were chosen because both genes were previously reported to be associated with the heart. Acadm is linked with cardiomyocyte differentiation and cold tolerance in mice [37]. Acadl is associated with sudden death occurring between 2 and 4 weeks of age after birth, with evidence of cardiomyopathy, as well as reduced litter sizes [36]. Additionally, genetic disorders of fatty acid oxidationrelated genes are known to exist in the human population and are recognized as important causes of morbidity and mortality [47]. 3.3.1. Acyl CoA dehydrogenase modulation Gender of the embryos was not addressed in this initial part of the study. We used RT-PCR analysis of E15.5 cardiac tissue from three mouse embryos from three different litters after the E6.75 acute exposures. All of the experimentally exposed mouse embryos displayed abnormal cardiac function. Acadl and Acadm expression was not greatly altered by Li or HCy exposure (Fig. 4A). Graphs of densitometric scan averages with standard deviations are shown to the right (Fig. 4B–E). Data was normalized in each case to the internal control ␤-actin. Although not statistically significant, a pattern of up-regulation of Acadm gene expression seemingly occurred in both Li+ - and HCy-exposure groups in comparison to control hearts (Fig. 4A). High FA supplementation maintained the expression close to control levels. We also analyzed gene expression a half a day after exposures in whole embryos at E7.5 and found that within 12 h, the changes in gene expression were observed (not shown). We next analyzed whether the gender of the embryo may relate to our results. Because evidence exists that abundance of mRNA may not correlate with that of protein [48], we also immunostained E15.5 embryonic male and female hearts, as well as placentas for the Acadm gene product, MCAD protein (Figs. 5 and 7 ). 3.3.1.1. MCAD protein expression in the heart. As seen in Fig. 5A–F and graphs 5G and 5H, in the male and female control hearts (Fig. 5A and D, respectively), a low level of MCAD protein is detectable in the right (RV) and left (LV) ventricular walls, in the trabecu-

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Fig. 6. Relative quantitation of Acadm gene expression in placental tissue using RT-PCR and taking into account gender: Results are shown for gene expression in placental tissue for Acadm (Fig. 6A; n = 3 embryos from three different litters for each treatment) and for Crt (Fig. 6B; n = 4 embryos). Embryos with Li+ /HCy exposures and control embryos are compared. Y-axis denotes average densitometric readings of gel scans; X-axis shows treatments: grey bars, male embryos; red bars, female embryos. Asterisks denote the p values for statistically significant treatments in comparison to control same gender group. A p value <0.05 is considered significant. * = p < 0.05; ** = p < 0.005; *** = p < 0.0005; “n” equals numbers of embryos.

lae, and in the interventricular septum (IVS; arrows). After acute HCy exposure, MCAD protein expression is highly elevated in the male heart (Fig. 5B; see also graph in Fig. 5G) with a lower level detectable in the female right and left ventricular walls and IVS (Fig. 5E, arrows). Lithium exposure suppressed MCAD expression in both male and female embryonic hearts (Fig. 5C and F), but a higher level remained evident in the female (see graph in Fig. 5H). In summary, male embryonic hearts in response to HCy displayed the most marked gender-associated change in MCAD protein expression. For the experimentally exposed hearts, the percentages of the HRP signal in the quantified regions in relation to the whole area are given for the delineated regions (the RV and LV myocardial walls and the IVS). Thus, male and female embryonic hearts of embryos from different litters responded differentially to Li+ and HCy exposures with male hearts being more affected with exposure to elevated HCy. 3.3.2. Modulation of acadm and MCAD expression in the placenta and association with embryonic gender After we had sorted our microarray lipid-related GO categories list by taking into account the sex of the embryos (Fig. 1A and B), it appeared possible that gender differences may relate to the results observed with ORO localization (Section 3.2.2) and with the initial RT-PCR analysis described above in Section 3.3.1. where the gender of the embryos had not been specified. We then tested for possible gender association by re-analyzing for Acadm gene expression in relation to the E15.5 placenta and specifying gender by RT-PCR genotyping (see Section 2, Section 2.6). We focused on lipid-related Acadm gene expression (Fig. 6A) and included Crt (calreticulin, Fig. 6B) that is not directly involved in lipid metabolism and determined whether a gender association would be noted. Crt is involved in calcium homeostasis and implantation during placental development [38]. RT-PCR analysis was carried out on samples of total RNA extracted from three male or three female placental tissues exposed to Li+ or to HCy and were compared to three male and three female

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Fig. 7. Gender-associated changes in MCAD protein expression in placental tissue on E15.5 after HCy- and Li+ − E6.75 exposures. Arrows point to some regions of localization. Male control placental tissue (Fig. 7A, B) compared with female control (Fig. 7G, H) and after exposures to HCy (male Fig. 7C,D; female 7I, J) and to Li+ (male Fig. 7E,F; female 7K,L). For each gender left column displays low magnification of the placenta and right-hand column higher magnification of a delineated region of the labyrinth. For gender comparisons in relation to HCy/Li+ exposures, the HRP signal was quantified in the maternal decidua and labyrinth and graph of data is shown in panel 7M. Abbreviations: MAT De, Maternal Decidua; Syn Tr, Syncytiotrophoblasts; Lab, labyrinth. Magnification bar in A for low 4× magnification = 1 mm; bar in B for 20× magnification = 100 ␮m. Fig. 7M shows quantitation of HRP signal expressed as a percentage of total field area analyzed. Solid grey bars depict male HCy-exposed tissue; solid red bars show female HCy- exposed tissue; patterned grey bar is male Li-exposed tissue; and patterned red is female Li+ −exposed tissue. LAB (on left), labyrinth tissue; MAT De (on right), maternal decidua. Inset in upper right in graph shows regions (green boxed-in areas) of placenta that were analyzed.

control placentas. All of these pregnant females received the health maintenance diet of 3.3 mg/kg. All of the treated embryos had been acutely exposed to either of the two factors and all displayed abnormal echo patterns, including semilunar valve defects. The Acadm expression changes were compared to control placental tissues of male and female embryos that displayed normal heart function. By taking into account gender and using ANOVA to analyze variance between the means of the groups and within groups, a statistical difference in expression at the 5% significance level (Pvalue equaled 0.007) was seen in response to HCy. Placental tissue of male embryos demonstrated a highly significant difference in Acadm expression level in response to HCy exposure than female embryos (Fig. 6A). ANOVA analysis indicated that a significant dif-

ference in Acadm expression between genders was not observed in the response to lithium. The calreticulin (Crt) gene displayed no gender association in its expression changes (Fig. 6B). Crt did show, however, a statistical difference between the groups at the 5% significance level, i.e. between control and experimental groups with both genders responding similarly in response to HCy or to Li+ exposure. MCAD protein expression in the placenta of male and female control embryos was detected in multiple cell types of the placenta, i.e., in the maternal decidua, syncytiotrophoblasts, and in the villi of the labyrinth layers (Fig. 7A). Quantification of HRP signal in gender-related maternal decidua and in labyrinth layers is shown as percentage of HRP signal in delineated regions in

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relation to the whole specific areas analyzed. Our description of levels of immunostaining is based on visual inspection of tissue and on software quantification of duplicate sections and shown in the graphs. The maternal decidua and labyrinth regions that were quantified are boxed-in the inset on graph. As observed, only a low level of localization was seen in the control labyrinth (Fig. 7B shown at higher magnification). Acute homocysteine early exposure resulted in an up-regulation of MCAD in the male placenta (Fig. 7C shown at low 4× magnification, 7D in labyrinth shown at higher 20× magnification) in contrast to the female placenta where a low level of expression remained in the maternal decidua (7I, arrows) similar to control tissue, and a low level of expression was noted in the labyrinth (7J). In response to Li+ exposure, the male placenta showed a down-regulation of MCAD in the maternal decidua (Fig. 7E) and little expression in the labyrinth (Fig. 7F); the female placenta also showed a down-regulation in the maternal decidua and displayed MCAD only in some areas in the labyrinth layer (arrows, Fig. 7L). In summary, the highest change in MCAD protein expression in the placenta was seen with male embryos in response to HCy and especially in the maternal decidua. A more modest increase in the labyrinth is also observed. For both genders little change was observed in response to lithium.

4. Discussion The bioinformatic results of the microarray data using GO categories indicated that lipid metabolism in cardiac tissue was primarily altered with folic acid deficiency, the latter exemplified by early embryonic exposure to an elevation of HCy. In addition, after sorting the defined GO categories list according to gender of the embryos compared with the various treatments versus control embryos, a gender bias appeared to exist in the response to the exposure with male embryos showing more lipid-related processes affected than in the female. We then validated the microarray-based results by demonstrating neutral lipid changes with exposures in the mouse heart and placenta using molecular and immunohistochemical techniques for changes in expression of Acyl CoA dehydrogenase medium length chain (Acadm) gene and its protein MCAD. We used Acadm as a biomarker because this gene appeared multiple times in the lipid-related GO categories that were altered and it is the most common inherited disorder of mitochondrial fatty acid ␤-oxidation in humans. Expression of Acadm and MCAD displayed significant changes in the placenta and in a gender-associated manner. We discuss below how lipid metabolism relates to cardiac and placental development and when dysregulated can lead to pathophysiology and birth defects. We then discuss how changes in lipids also can alter Wnt signaling that is impacted more directly by the Li+ exposure. Lastly we describe the intersection of lipid metabolism with the folic acid cycle.

4.1. Lipid metabolism and disease Bioenergetic maturation has been demonstrated to be an important component of normal cardiomyocyte differentiation [49]. As the developing heart increasingly relies on oxidative metabolism, as the vasculature brings blood to the heart from the placenta, there is a higher demand for energy to maintain continuous myocardial contractile activity. This energy requirement is primarily met by ß-oxidation of fatty acids in the mitochondria. Myocardial fatty acid metabolism in health and disease has been reviewed in detail [50]. Recent evidence demonstrates that cytoplasmic lipases remove fatty acids from lipid droplets when cells are starved and enable their transfer into mitochondria [51]. By E14

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mouse cardiac mitochondria are engaged in oxidative phosphorylation and fatty acid oxidation has been initiated [49]. Perturbation of placental development can lead to altered blood flow, smaller placentas, growth-restricted embryos, and to resorption of embryos that we also observed in our published studies [3,11]. With the exception of the perfusing maternal blood, the placenta is an embryonic tissue [52]. The placenta is composed of trophoblast cells originating from the trophectoderm of the blastocyst. Between embryonic days E4.5 and E7.5 in the mouse model, the differentiation of different trophoblast lineages is ongoing [53]. Our exposures at E6.75 thus target this time-period of differentiation. Subsequently, the extra-embryonic ectoderm expands and forms the chorionic epithelium. Posteriorly the allantois forms from the embryonic mesoderm and contacts the chorion at ∼E8.5. Fetoplacental blood vessels later grow in from the allantois to form the fetal components of the vascular network of the placental labyrinth. The labyrinth is where nutrient and gas exchange occurs between the maternal and fetal circulations. Thus, perturbation of trophoblast differentiation early during gestation has the potential to alter subsequent placental organization and function. As we demonstrated, Li+ and HCy exposures on E6.75 resulted in disorganization of the villi and placental lipid changes in the labyrinth and maternal decidual layers with large areas of these regions devoid of neutral lipids, as well as displaying altered MCAD expression. The control placenta shows a high level of lipid droplets accumulated in cells with a normal nutrient supply. Possibly under stress or with nutrient deprivation as with folic acid deficiency, the placenta breaks down lipids to enable their transfer to the embryo to maintain embryonic development and growth. Abnormal or reduced fatty acid transfer during development would alter the fatty acid composition of tissue lipids having short- and long-term effects on cell structure and function, including cell signaling, and can contribute subsequently to cardiac pathology. A mismatch in the adult heart of fatty acid uptake/utilization, for example, leads to an accumulation of lipids that can be toxic to cardiac myocytes leading to ventricular dysfunction and premature death. In the embryo such a mismatch could result in suboptimal nutrition, oxygenation, and alterations in the bioenergetics of the cells resulting in decreased embryonic myocardial performance [3,16,54]. An importance of the placenta-heart axis for heart development was recently reviewed [55,56]. As demonstrated in the results, the fatty acid oxidation gene Acadm and its protein MCAD are modulated in the heart and placenta with exposures to Li+ and HCy. Addressing gender differences in gene expression in the heart and placenta, it was observed that with an elevation of HCy, a hallmark of nutrient deficiency, male embryos showed a statistically significant upregulation of Acadm expression in comparison to females in which Acadm remained close to control levels. The up-regulation may be a response to compensate for the nutrient deficiency. In the human population genetic disorders of mitochondrial fatty acid-oxidation have been recognized as important causes of morbidity and mortality, indicating the physiological significance of fatty acids in general for cellular energy production during periods of fasting and metabolic stress. Human MCAD (ACADM) deficiency is the most frequently encountered disorder of the fatty acid oxidation pathway and overall is one of the most recognizable inborn errors of metabolism [47]. Mouse models have been generated for several of the enzymes involved in fatty acid oxidation to analyze underlying causes for the disease characteristics. In general the mouse models of disorders of mitochondrial fatty acid beta-oxidation have shown clinical signs that include Reye-like syndrome and cardiomyopathy, and many are cold intolerant [57]. Extrapolating our results to the first month of human pregnancy, modulation of fatty acid oxidation-related enzymes such as Acadm by even mild folate deficiency that elevates HCy levels and in the context of possible inborn errors of

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metabolism as for example, MTHFR allele heterozygosity [45,46], it could lead to more pronounced metabolic changes that manifest in the offspring in late fetal stages or after birth and, as shown in our analysis, the severity may have a gender relationship with male embryos being more affected. Our analysis may provide a basis for certain adult cardiovascular diseases as well, based on the theory of developmental origins of adult diseases [58,59]. 4.2. Dyslipidemia and birth defects Based on ORO localization, neutral lipid droplet distribution was altered more in the placenta than in the heart. Despite the lack of a mechanism specified in published studies, investigations have indicated that maternal dyslipidemia during human pregnancy increases the risk of adverse pregnancy outcomes and congenital heart defects [60,61]. A similar fetal metabolic effect exists in association with maternal diabetes [62]. 4.3. Lipids and Wnt signaling Besides energy storage, lipids serve in two other critical functions by being structural components of membranes in lipid rafts and by lipid modifications of proteins regulating activity of cell signaling factors. Lipid rafts are dynamic subdomains of the plasma membrane that contain high concentrations of cholesterol and glycosphingolipids and have an important role in signal transduction [63,64]. Many cell signaling proteins localize into the lipid raft domains of the plasma membrane. It was reported that cholesterol levels, a major component of lipid rafts, could affect gene expression during skeletal muscle differentiation [65]. Besides that of Wnt signaling, cholesterol levels were reported also to modulate other growth factor signaling, e.g., EGF receptor-mediated, signaling [66]. Lipid modifications are known to be necessary for active Wnt signaling [67,68] via two fatty acid modifications [69] implicated in its secretion. After lipid modification, Drosophila DWnt-1 partitions as a membrane-anchored protein and is sorted into lipid raft detergent-insoluble microdomains of the plasma membrane [70]. Interestingly, an elevation of HCy was reported to significantly increase glomerular endothelial cell permeability by stimulating lipid raft clustering to form redox signaling platforms [71]. Lipids are thus critical in fetal development, not only as components of membrane rafts and phospholipids, but also as ligands for receptors and transcription factors in gene regulation and in direct interactions with proteins. Thus, Li+ that directly inhibits a critical step in the Wnt signaling pathway, GSK-3␤ activity [72], has the potential similarly to HCy, the latter through lipid changes related to Wnt activity, to affect early cardiomyocyte and trophoblast differentiation and normal tissue formation downstream. It appears that further study of placental lipid changes with embryonic exposures during early pregnancy is an important area for future study in the effects on early development of the embryo and possibly for later adult cardiovascular diseases [58,59,73]. 4.4. Intersection of lipid and folic acid metabolism The intersection of lipid and folic acid metabolism suggest that epigenetic regulation also is involved, as the folic acid cycle leads to formation of S-adenosylmethionine (SAM) the primary methyl donor for epigenetic modifications. It is apparent that the metabolic or nutritional state of the organism directly influences epigenetic modifications: Epigenetic modifications rely on substrates of intermediary metabolism such as SAM, acetyl CoA, ␣-ketoglutarate and nicotinamide adenine dinucleotide [74]. Because the same signaling pathways active in cardiogenesis are involved in placentation, neurogenesis, neural crest differentiation, and craniofacial development, the development of multiple tissues can be affected during

gestation. As an example, children with congenital heart defects are reported to be at increased risk of neural developmental disorders or disabilities, or developmental delay [75]. The severity of defects and those that predominate would reflect timing of exposures during gestation, the level of the exposure, and as based on our present results, possibly gender. A commonality of pathways appears to be emerging that result in cardiac birth defects induced by multiple factors that link folic acid deficiency with altered lipid metabolism. These results may relate also to the cardiac defects that are observed with dyslipidemia reported in association with pregnancies involving alcohol abuse and result in Fetal Alcohol Spectrum of Defects (FASD). In addition maternal diabetes and obesity are conditions that dysregulate lipid metabolism and are associated with congenital heart defects [76–80]. The folic acid pathway and above-mentioned disease states are interrelated also with Wnt signaling. Wnt signaling is linked with adipocyte cell lineage and obesity [81], and with folic acid metabolism in the mouse embryo [82], whereby folic acid supplementation normalizes hyperactive WNT activity. These intersecting pathways are involved during early steps of cell differentiation during placentation and embryogenesis in human pregnancy, and when lipids are dysregulated, an association with congenital heart defects in the offspring is observed [60]. A means of prevention of the birth defects induced by dyslipidemia appears to exist by administering a higher folate supplementation dose than currently formulated in prenatal vitamin preparations and that the supplementation is initiated periconceptionally. The necessary safe and effective dose for prevention of cardiac defects in human pregnancy requires further clinical and epidemiological studies [83], although we used the 10.5 mg/kg dose based on a published clinical study indicating this level had been shown to be effective for prevention of birth defects in human pregnancy [28]. It was stated recently in the Folate Expert Panel review paper that there does not appear to be any toxic or abnormal effects of circulating plasma folic acid [84]. Epidemiological, clinical and animal studies taken together demonstrate that the intrauterine environment influences growth and development of the embryo and fetus. Our results using the mouse model, when extrapolated to human pregnancy, demonstrate that in the first month, specifically in the second and third week post-conception, the human embryo is highly vulnerable to exposure to environmental factors encountered by the pregnant female. This critical window that we targeted coincides with a period of cell specification and differentiation of cardiomyocytes and trophoblast cells. Thus, an early intrauterine insult before a woman recognizes her pregnancy already may have long-lasting effects on tissue and organ function during pregnancy and postnatally.

5. Conclusions Bioinformatic analysis of microarray data of embryonic heart tissue, with and without high dose folic acid supplementation during gestation, generated a list of common GO categories that demonstrated that with Li+ and HCy embryonic exposures, a high number of the affected categories related to lipid metabolism and more alterations were observed in the male embryonic heart transcriptome in relation to HCy exposure than in the female. Validation of lipid changes was observed with Oil Red O-staining for neutral lipid localization in embryonic mouse embryos demonstrating that lipid droplets were altered with early Li+ and HCy exposures in the fetal E15.5 four-chambered heart and in the placenta with greater changes occurring in placental tissue. Modulation of the fatty acid oxidation gene Acyl CoA Dehydrogenase Medium Length Chain (Acadm) and its protein MCAD occurs in the heart and pla-

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centa with exposures. A gender bias to the gene modulation in response to HCy exposure existed with placental tissue of male embryos demonstrating a highly significant difference in Acadm expression as compared to female embryos and controls. A gender association for a higher level of MCAD protein expression was also noted in the male embryonic heart in response to HCy. The gender bias we suggest may relate to the increased severity of heart defects observed in male children, when compared to the female in the human population. High dose folic acid dietary supplementation on morning of conception can prevent or reduce the above changes in neutral lipids and Acadm/MCAD expression in cardiac and placental tissues in both male and female embryos. Transparency document The Transparency document associated with this article can be found in the online version. Funding The support from Suncoast Cardiovascular Research and Education Foundation founded by Helen Harper Brown (KKL) and the David and Janice Mason Foundation (KKL) of the USF Morsani College of Medicine for these studies is gratefully acknowledged. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.reprotox.2016. 03.039. References [1] Global report on birth defects, The Hidden Toll of Dying and Disabled Children, March of Dimes, New York, USA: White Plains, 2006, p. p. 28. [2] J. Chen, M. Han, S.M. Manisastry, P. Trotta, M.C. Serrano, J.C. Huhta, et al., Molecular effects of lithium exposure during mouse and chick gastrulation and subsequent valve dysmorphogenesis, Birth Defects Res. A Clin. Mol. Teratol. 82 (2008) 508–518. [3] M. Han, M.C. Serrano, R. Lastra-Vicente, P. Brinez, G. Acharya, J.C. Huhta, et al., Folate rescues lithium-, homocysteine- and Wnt3A-induced vertebrate cardiac anomalies, Dis. Model Mech. 2 (2009) 467–478. [4] S.M. Manisastry, M. Han, K.K. Linask, Early temporal-specific responses and differential sensitivity to lithium and Wnt-3A exposure during heart development, Dev. Dyn. 235 (2006) 2160–2174. [5] D. Walker, C. Fisher, A. Sherman, B. Wybrecht, K. Kyndely, Fetal alcohol spectrum disorders prevention: an exploratory study of women’s use of, attitudes toward, and knowledge about alcohol, J. Am. Acad. Nurse Pract. 17 (2005) 187–193. [6] J.S. Fitzgerald, A. Germeyer, B. Huppertz, U. Jeschke, M. Knofler, G. Moser, et al., Governing the invasive trophoblast: current aspects on intra- and extracellular regulation, Am. J. Reprod. Immunol. 63 (2010) 492–505. [7] S.J. Monkley, S.J. Delaney, D.J. Pennisi, J.H. Christiansen, Targeted disruption of the Wnt2 gene results in placentation defects, Development 122 (1996) 3343–3353. [8] S. Peng, J. Li, C. Miao, L. Jia, Z. Hu, P. Zhao, et al., Dickkopf-1 secreted by decidual cells promotes trophoblast cell invasion during murine placentation, Reproduction 135 (2008) 367–375. [9] S. Sonderegger, P. Haslinger, A. Sabri, C. Leisser, J.V. Otten, C. Fiala, et al., Wingless (Wnt)-3A induces trophoblast migration and matrix metalloproteinase-2 secretion through canonical Wnt signaling and protein kinase B/AKT activation, Endocrinology 151 (2010) 211–220. [10] S. Sonderegger, J. Pollheimer, M. Knofler, Wnt signalling in implantation, decidualisation and placental differentiation—review, Placenta 31 (2010) 839–847. [11] M. Han, A.L. Neves, M. Serrano, P. Brinez, J.C. Huhta, G. Acharya, et al., Effects of alcohol, lithium, and homocysteine on nonmuscle myosin-II in the mouse placenta and human trophoblasts, Am. J. Obstet. Gynecol. 207 (140) (2012) e7–e19. [12] A.P. Brandalize, E. Bandinelli, P.A. dos Santos, I. Roisenberg, L. Schuler-Faccini, Evaluation of C677T and A1298C polymorphisms of the MTHFR gene as maternal risk factors for down syndrome and congenital heart defects, Am. J. Med. Genet. A 149A (2009) 2080–2087. [13] E. Goldmuntz, S. Woyciechowski, D. Renstrom, P.J. Lupo, L.E. Mitchell, Variants of folate metabolism genes and the risk of conotruncal cardiac defects, Circ. Cardiovasc. Genet. 1 (2008) 126–132.

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